Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

We report the synthesis of two C4′-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4′-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.Laboratorium voor Medicinale chemie.status: publishe

A Purpose-Built System for Culturing Cells as <em>In Vivo</em> Mimetic 3D Structures

Culturing cells in 3D is often considered to be significantly more difficult than culturing them in 2D. In practice, this is not the case: the situation is that equipment needed for 3D cell culture has not been optimised as much as equipment for 2D. Here we present a few key features which must be considered when designing 3D cell culture equipment. These include diffusion gradients, shear stress and time. Diffusion gradients are unavoidably introduced when cells are cultured as clusters. Perhaps the most important consequence of this is that the resulting hypoxia is a major driving force in the metabolic reprogramming. Most cells in tissues do not experience liquid shear stress and it should therefore be minimised. Time is the factor that is most often overlooked. Cells, irrespective of their origin, are damaged when cultures are initiated: they need time to recover. All of these features can be readily combined into a clinostat incubator and bioreactor. Surprisingly, growing cells in a clinostat system do not require specialised media, scaffolds, ECM substitutes or growth factors. This considerably facilitates the transition to 3D. Most importantly, cells growing this way mirror cells growing in vivo and are thus valuable for biomedical research

Transcription factor decoy oligonucleotides modified with locked nucleic acids: an in vitro study to reconcile biostability with binding affinity

Double-stranded oligonucleotides (ODNs) containing the consensus binding sequence of a transcription factor provide a rationally designed tool to manipulate gene expression at the transcriptional level by the decoy approach. However, modifications introduced into oligonucleotides to increase stability quite often do not guarantee that transcription factor affinity and/or specificity of recognition are retained. We have previously evaluated the use of locked nucleic acids (LNA) in the design of decoy molecules for the transcription factor κB. Oligo nucleotides containing LNA substitutions displayed high resistance to exo- and endonucleolytic degradation, with LNA–DNA mix-mers being more stable than LNA–DNA–LNA gap-mers. However, insertion of internal LNA bases resulted in a loss of affinity for the transcription factor. This latter effect apparently depended on positioning of the internal LNA substitutions. Indeed, here we demonstrate that intra- and inter-strand positioning of internal LNAs has to be carefully considered to maintain affinity and achieve high stability, respectively. Unfortun ately, our data also indicate that LNA positioning is not the only parameter affecting transcription factor binding, the interference in part being dependent on the intrinsic conformational properties of this nucleotide analog. To circumvent this problem, the successful use of an α-l-ribo- configured LNA is demonstrated, indicating LNA–DNA–α-l-LNA molecules as promising new decoy agents